Cultivating complexity: Advancements in establishing in vitro models for the mucus-adhering gut microbiota.

A healthy mucus is essential for maintaining intestinal homeostasis and overall well-being. In recent years, extensive research focused on understanding the intricate interactions between mucus and the gut microbiota. Mucus-adhering bacteria play crucial roles in preserving barrier integrity, epithelial permeability and mucus architecture, as well as in the colonization resistance against pathogens. Unravelling the significance of these microorganisms in human health and disease is challenging, primarily because most of the studies on the human gut microbiota rely on faecal samples, which do not fully represent the microecological complexity found in the intestinal mucosa. This review discusses novel strategies to specifically target and evaluate the mucosal microbiota, such as culturomics applied to mucosal biopsies or brushings, intestinal organoids and artificial in vitro models incorporating mucus.

Dissecting the role of the MS-ring protein FliF in Bacillus cereus flagella-related functions.

The flagellar MS-ring, uniquely constituted by FliF, is essential for flagellar biogenesis and functionality in several bacteria. The aim of this study was to dissect the role of FliF in the Gram-positive and peritrichously flagellated Bacillus cereus. We demonstrate that fliF forms an operon with the upstream gene fliE. In silico analysis of B. cereus ATCC 14579 FliF identifies functional domains and amino acid residues that are essential for protein functioning. The analysis of a ΔfliF mutant of B. cereus, constructed in this study using an in frame markerless gene replacement method, reveals that the mutant is unexpectedly able to assemble flagella, although in reduced amounts compared to the parental strain. Nevertheless, motility is completely abolished by fliF deletion. FliF deprivation causes the production of submerged biofilms and affects the ability of B. cereus to adhere to gastrointestinal mucins. We additionally show that the fliF deletion does not compromise the secretion of the three components of hemolysin BL, a toxin secreted through the flagellar type III secretion system. Overall, our findings highlight the important role of B. cereus FliF in flagella-related functions, being the protein required for complete flagellation, motility, mucin adhesion, and pellicle biofilms.

Anti-staphylococcal activity of a polyphenol-rich citrus extract: synergy with ß-lactams and low proficiency to induce resistance.

Introduction: Antibiotic resistance represents one of the most significant threats to public health in the 21st century. Polyphenols, natural molecules with antibacterial activity produced by plants, are being considered as alternative antimicrobial strategies to manage infections caused by drug-resistant bacteria. In this study, we investigated the antibacterial activity of a polyphenol mixture extracted from citrus fruits, against both antibiotic-susceptible and resistant strains of Staphylococcus aureus and Staphylococcus epidermidis. Methods: Broth microdilution and time-kill curve experiments were used to test the extract anti-staphylococcal activity. Cytotoxicity was assessed by the hemolysis assay. The interaction between the mixture and antibiotics was investigated by the checkerboard assay. The effect of B alone and in combination with oxacillin on the membrane potential was investigated by the 3,3'-dipropylthiadicarbocyanine iodide assay. The ability of the extract to induce the development of resistance was verified by propagating S. aureus for 10 transfers in the presence of sub-inhibitory concentrations. Results: The citrus extract was found to be active against all Staphylococcus strains at remarkably low concentrations (0.0031 and 0.0063%), displaying rapid bactericidal effects without being toxic on erythrocytes. In particular, B was found to rapidly cause membrane depolarization. When combined with methicillin, meropenem, and oxacillin, the mixture displayed synergistic activity exclusively against methicillin-resistant strains. We additionally show that the sequential exposure of S. aureus to sub-inhibitory concentrations did not induce the development of resistance against the extract. Discussion: Overall, these findings support the potential use of the citrus extract as promising option to manage staphylococcal infections and suggest that it may counteract the mechanism behind methicillin-resistance.

In-depth microbiological characterization of urine from subjects with type 2 diabetes.

CONTEXT: Lower urinary tract symptoms (LUTS) are common in type 2 diabetes (T2D), affecting quality of life and potentially leading to medication discontinuation. Among various factors contributing to LUTS, recent observations suggest a critical role of the urinary microbiota. Research on urinary dysbiosis in T2D remains underexplored. OBJECTIVE: We conducted a pilot study to investigate differences in the urinary microbiota between T2D patients and healthy individuals and its potential indirect association with LUTS risk. METHODS: This case-control study included 50 patients with T2D and no LUTS, and 25 healthy controls. Microbial DNAs were extracted from urinary sediments and bacterial populations quantified by Real-Time qPCR and qualitatively investigated by 16S rRNA gene sequencing. Validation experiments with Digital PCR were also performed. RESULTS: In T2D patients a higher total bacterial load and an increased abundance of Bacillota were found. After stratification by gender, these results were observed only in women. However, no significant quantitative differences were observed at the genus level. Alpha diversity analysis showed no significant differences between T2D and control groups, or by gender. At the species level, a substantial qualitative and often gender-dependent shift was present in T2D individuals. CONCLUSIONS: The urinary microbiome of subjects with T2D was found to be different from that of healthy controls. Specifically, T2D patients displayed higher total bacterial load and Bacillota levels, as well as qualitative changes in bacterial species. These changes suggested a dysbiotic condition of the urinary microbiota of T2D subjects, with some gender-related differences. Although causality cannot be inferred, these findings highlight the impact of T2D on the urinary microbiota and its potential relevance in developing LUTS and, from a broader perspective, metabolic abnormalities.

Anti-Staphylococcal Biofilm Effects of a Liposome-Based Formulation Containing Citrus Polyphenols.

Biofilms are surface-associated microbial communities embedded in a matrix that is almost impenetrable to antibiotics, thus constituting a critical health threat. Biofilm formation on the cornea or ocular devices can lead to serious and difficult-to-treat infections. Nowadays, natural molecules with antimicrobial activity and liposome-based delivery systems are proposed as anti-biofilm candidates. In this study, the anti-biofilm activity of a formulation containing citrus polyphenols encapsulated in liposomes was evaluated against Staphylococcus aureus and Staphylococcus epidermidis, the most common agents in ocular infections. The formulation activity against planktonic staphylococci was tested by broth microdilution and sub-inhibitory concentrations were used to evaluate the effect on biofilm formation using the crystal violet (CV) assay. The eradicating effect of the preparation on mature biofilms was investigated by the CV assay, plate count, and confocal laser scanning microscopy. The product was bactericidal against staphylococci at a dilution of 1:2 or 1:4 and able to reduce biofilm formation even if diluted at 1:64. The formulation also had the ability to reduce the biomass of mature biofilms without affecting the number of cells, suggesting activity on the extracellular matrix. Overall, our results support the application of the used liposome-encapsulated polyphenols as an anti-biofilm strategy to counter biofilm-associated ocular infections.

A Millifluidic Chamber for Controlled Shear Stress Testing: Application to Microbial Cultures.

In vitro platforms such as bioreactors and microfluidic devices are commonly designed to engineer tissue models as well as to replicate the crosstalk between cells and microorganisms hosted in the human body. These systems promote nutrient supply and waste removal through culture medium recirculation; consequently, they intrinsically expose cellular structures to shear stress, be it a desired mechanical stimulus to drive the cell fate or a potential inhibitor for the model maturation. Assessing the impact of shear stress on cellular or microbial cultures thus represents a crucial step to define proper environmental conditions for in vitro models. In this light, the aim of this study was to develop a millifluidic device enabling to generate fully controlled shear stress profiles for quantitatively probing its influence on tissue or bacterial models, overcoming the limitations of previous reports proposing similar devices. Relying on this millifluidic tool, we present a systematic methodology to test how adherent cellular structures react to shear forces, which was applied to the case of microbial biofilms as a proof of concept. The results obtained suggest our approach as a suitable testbench to evaluate culture conditions in terms of shear stress faced by cells or microorganisms.

Impact of Bacillus cereus on the Human Gut Microbiota in a 3D In Vitro Model.

In vitro models for culturing complex microbial communities are progressively being used to study the effects of different factors on the modeling of in vitro-cultured microorganisms. In previous work, we validated a 3D in vitro model of the human gut microbiota based on electrospun gelatin scaffolds covered with mucins. The aim of this study was to evaluate the effect of Bacillus cereus, a pathogen responsible for food poisoning diseases in humans, on the gut microbiota grown in the model. Real-time quantitative PCR and 16S ribosomal RNA-gene sequencing were performed to obtain information on microbiota composition after introducing B. cereus ATCC 14579 vegetative cells or culture supernatants. The adhesion of B. cereus to intestinal mucins was also tested. The presence of B. cereus induced important modifications in the intestinal communities. Notably, levels of Proteobacteria (particularly Escherichia coli), Lactobacillus, and Akkermansia were reduced, while abundances of Bifidobacterium and Mitsuokella increased. In addition, B. cereus was able to adhere to mucins. The results obtained from our in vitro model stress the hypothesis that B. cereus is able to colonize the intestinal mucosa by stably adhering to mucins and impacting intestinal microbial communities as an additional pathogenetic mechanism during gastrointestinal infection.

Development of an In Vitro Model of the Gut Microbiota Enriched in Mucus-Adhering Bacteria.

Culturing the gut microbiota in in vitro models that mimic the intestinal environment is increasingly becoming a promising alternative approach to study microbial dynamics and the effect of perturbations on the gut community. Since the mucus-associated microbial populations in the human intestine differ in composition and functions from their luminal counterpart, we attempted to reproduce in vitro the microbial consortia adhering to mucus using an already established three-dimensional model of the human gut microbiota. Electrospun gelatin structures supplemented or not with mucins were inoculated with fecal samples and compared for their ability to support microbial adhesion and growth over time, as well as to shape the composition of the colonizing communities. Both scaffolds allowed the establishment of long-term stable biofilms with comparable total bacterial loads and biodiversity. However, mucin-coated structures harbored microbial consortia especially enriched in Akkermansia, Lactobacillus, and Faecalibacterium, being therefore able to select for microorganisms commonly considered mucosa-associated in vivo. IMPORTANCE These findings highlight the importance of mucins in shaping intestinal microbial communities, even those in artificial gut microbiota systems. We propose our in vitro model based on mucin-coated electrospun gelatin structures as a valid device for studies evaluating the effects of exogenous factors (nutrients, probiotics, infectious agents, and drugs) on mucus-adhering microbial communities.

Analysis of the microbial content of probiotic products commercialized worldwide and survivability in conditions mimicking the human gut environment.

Introduction: Probiotics are living microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Adequate number of living microbes, the presence of specific microorganisms, and their survival in the gastrointestinal (GI) environment are important to achieve desired health benefits of probiotic products. In this in vitro study, 21 leading probiotic formulations commercialized worldwide were evaluated for their microbial content and survivability in simulated GI conditions. Methods: Plate-count method was used to determine the amount of living microbes contained in the products. Culture-dependent Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry and culture-independent metagenomic analysis through 16S and 18S rDNA sequencing were applied in combination for species identification. To estimate the potential survivability of the microorganisms contained in the products in the harsh GI environment, an in vitro model composed of different simulated gastric and intestinal fluids was adopted. Results: The majority of the tested probiotic products were concordant with the labels in terms of number of viable microbes and contained probiotic species. However, one product included fewer viable microbes than those displayed on the label, one product contained two species that were not declared, and another product lacked one of the labeled probiotic strains. Survivability in simulated acidic and alkaline GI fluids was highly variable depending on the composition of the products. The microorganisms contained in four products survived in both acidic and alkaline environments. For one of these products, microorganisms also appeared to grow in the alkaline environment. Conclusion: This in vitro study demonstrates that most globally commercialized probiotic products are consistent with the claims described on their labels with respect to the number and species of the contained microbes. Evaluated probiotics generally performed well in survivability tests, although viability of microbes in simulated gastric and intestinal environments showed large variability. Although the results obtained in this study indicate a good quality of the tested formulations, it is important to stress that stringent quality controls of probiotic products should always be performed to provide optimal health benefits for the host.

HPLC-MS-MS quantification of short-chain fatty acids actively secreted by probiotic strains.

Introduction: Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host's physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFA deficiency. Methods: In this research, nine probiotic strains (Bacillus clausii NR, OC, SIN, and T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS). Results: The developed method was highly sensitive and specific, showing excellent limits of detection and quantification of secreted SCFAs. All tested microorganisms were shown to secrete acetic acid, with only B. clausii and S. boulardii additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced. Discussion: The experimental approach described in this study may contribute to the characterization of probiotics as SCFA-producing organisms, a crucial stage toward their application to improve SCFA deficiency.

Prevalence, Virulence Potential, and Growth in Cheese of Bacillus cereus Strains Isolated from Fresh and Short-Ripened Cheeses Sold on the Italian Market.

This study investigated B. cereus presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market. B. cereus was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different B. cereus strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (sph, plcA, cytK, entFM, bcet, nheA, nheB, nheC). Overall, 92% of strains harbored bcet, 75% the three genes nheA, nheB, and nheC, as well as plcA and sph, 67% entFM, and 33% cytK. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for B. cereus growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.

Animal and In Vitro Models as Powerful Tools to Decipher the Effects of Enteric Pathogens on the Human Gut Microbiota.

Examining the interplay between intestinal pathogens and the gut microbiota is crucial to fully comprehend the pathogenic role of enteropathogens and their broader impact on human health. Valid alternatives to human studies have been introduced in laboratory practice to evaluate the effects of infectious agents on the gut microbiota, thereby exploring their translational implications in intestinal functionality and overall health. Different animal species are currently used as valuable models for intestinal infections. In addition, considering the recent advances in bioengineering, futuristic in vitro models resembling the intestinal environment are also available for this purpose. In this review, the impact of the main human enteropathogens (i.e., Clostridioides difficile, Campylobacter jejuni, diarrheagenic Escherichia coli, non-typhoidal Salmonella enterica, Shigella flexneri and Shigella sonnei, Vibrio cholerae, and Bacillus cereus) on intestinal microbial communities is summarized, with specific emphasis on results derived from investigations employing animal and in vitro models.

In vitro assessment of probiotic attributes for strains contained in commercial formulations.

Although probiotics are often indiscriminately prescribed, they are not equal and their effects on the host may profoundly differ. In vitro determination of the attributes of probiotics should be a primary concern and be performed even before clinical studies are designed. In fact, knowledge on the biological properties a microbe possesses is crucial for selecting the most suitable bacteriotherapy for each individual. Herein, nine strains (Bacillus clausii NR, OC, SIN, T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) declared to be contained in six commercial formulations were tested for their ability to tolerate simulated intestinal conditions, adhere to mucins, and produce ß-galactosidase, antioxidant enzymes, riboflavin, and D-lactate. With the exception of B. breve, all microbes survived in simulated intestinal fluid. L. rhamnosus was unable to adhere to mucins and differences in mucin adhesion were evidenced for L. reuteri and S. boulardii depending on oxygen levels. All microorganisms produced antioxidant enzymes, but only B. clausii, B. coagulans, B. breve, and L. reuteri synthesize ß-galactosidase. Riboflavin secretion was observed for Bacillus species and L. rhamnosus, while D-lactate production was restricted to L. reuteri and L. rhamnosus. Our findings indicate that the analyzed strains possess different in vitro biological properties, thus highlighting the usefulness of in vitro tests as prelude for clinical research.

Bacillus cereus in Dairy Products and Production Plants.

Spore-forming Bacillus cereus is a common contaminant of dairy products. As the microorganism is widespread in the environment, it can contaminate milk at the time of milking, but it can also reach the dairy products in each phase of production, storage and ripening. Milk pasteurization treatment is not effective in reducing contamination and can instead act as an activator of spore germination, and a potential associated risk still exists with the consumption of some processed foods. Prevalences and concentrations of B. cereus in milk and dairy products are extremely variable worldwide: in pasteurized milk, prevalences from 2% to 65.3% were reported, with concentrations of up to 3 × 105 cfu/g, whereas prevalences in cheeses ranged from 0 to 95%, with concentrations of up to 4.2 × 106 cfu/g. Bacillus cereus is also well known to produce biofilms, a serious concern for the dairy industry, with up to 90% of spores that are resistant to cleaning and are easily transferred. As the contamination of raw materials is not completely avoidable, and the application of decontamination treatments is only possible for some ingredients and is limited by both commercial and regulatory reasons, it is clear that the correct application of hygienic procedures is extremely important in order to avoid and manage the circulation of B. cereus along the dairy supply chain. Future developments in interventions must consider the synergic application of different mild technologies to prevent biofilm formation and to remove or inactivate the microorganism on the equipment.

Characterization of a Bacillus cereus strain associated with a large feed-related outbreak of severe infection in pigs.

AIMS: Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. METHODS AND RESULTS: Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. CONCLUSIONS: The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements.

Study of the Adhesion of the Human Gut Microbiota on Electrospun Structures.

Although the adhesion of bacteria on surfaces is a widely studied process, to date, most of the works focus on a single species of microorganisms and are aimed at evaluating the antimicrobial properties of biomaterials. Here, we describe how a complex microbial community, i.e., the human gut microbiota, adheres to a surface to form stable biofilms. Two electrospun structures made of natural, i.e., gelatin, and synthetic, i.e., polycaprolactone, polymers were used to study their ability to both promote the adhesion of the human gut microbiota and support microbial growth in vitro. Due to the different wettabilities of the two surfaces, a mucin coating was also added to the structures to decouple the effect of bulk and surface properties on microbial adhesion. The developed biofilm was quantified and monitored using live/dead imaging and scanning electron microscopy. The results indicated that the electrospun gelatin structure without the mucin coating was the optimal choice for developing a 3D in vitro model of the human gut microbiota.

The administration of Enterococcus faecium SF68 counteracts compositional shifts in the gut microbiota of diet-induced obese mice.

Microorganisms with probiotic properties are eliciting an increasing interest as coadjuvants in the prevention and treatment of obesity through modulation of the gut microbiota. In this study, a probiotic formulation based on Enterococcus faecium SF68 was administered to mice fed with a high-fat diet (HFD) to evaluate its efficacy in reducing body mass gain and in modulating the intestinal bacterial composition. Both stool and ileum samples were collected from untreated and treated mice and absolute abundances of specific taxa constituting the gut microbial consortium were evaluated. SF68 administration significantly reduced the HFD-induced weight gain. In these animals, the microbial gut composition shifted toward an enrichment in microbes positively correlated with mucus thickness, lower inflammation, lower glycemia levels, and SCFA production (i.e., Bifidobacterium, Akkermansia, and Faecalibacterium), as well as a depletion in bacterial phyla having a key role in obesity (i.e., Firmicutes, Proteobacteria). Our results demonstrate the efficacy of E. faecium SF68 in adjusting the composition of the dysbiotic microbiota of HFD-fed animals, thus ameliorating clinical conditions and exerting anti-obesity effects.

Corrigendum: Compositional quality and potential gastrointestinal behavior of probiotic products commercialized in Italy.

[This corrects the article DOI: 10.3389/fmed.2018.00059.].

Antimicrobial Activity of Xibornol and a Xibornol-Based Formulation Against Gram-Positive Pathogens of the Respiratory Tract.

Xibornol is known since the 70s and a xibornol-based formulation is commercialized as spray suspension for the antisepsis of the oral cavity and as adjuvant in pharyngeal infections caused by Gram-positive microorganisms. Herein, we evaluated the antimicrobial activity of xibornol and the xibornol-based formulation against common pathogens of the upper and lower respiratory tract.Our results indicate that xibornol alone and the xibornol-based formulation have strong antibacterial action against Streptococcus pneumoniae, Streptococcus pyogenes, and Staphyloccus aureus, as well as against the two emerging pathogens Actinomyces israelii and Corynebacterium ulcerans. These findings highlight the antimicrobial potential of these drugs in the topical control of pathogenic Gram-positive bacteria of the respiratory tract.

Microbiological Quality and Resistance to an Artificial Gut Environment of Two Probiotic Formulations.

The quality control of probiotic products is the focus of numerous organizations worldwide. Several studies have highlighted the poor microbiological quality of many commercial probiotic formulations in terms of the identity of the contained microorganisms, viability, and purity, thus precluding the expected health benefits and representing a potential health risk for consumers. In this paper, we analyzed the contents of two probiotic formulations, one composed of an encapsulated mixture of lactobacilli and bifidobacteria, and one by a lyophilized yeast. The microorganisms contained in the products were quantified and identified using up-to-date methodologies, such as MALDI-TOF MS and metagenomic analysis. Moreover, as acid and bile tolerance is included among the criteria used to select probiotic microorganisms, in vitro tests were performed to evaluate the behavior of the formulations in conditions mimicking the harsh gastric environment and the intestinal fluids. Our results indicate the high quality of the formulations in terms of the enumeration and identification of the contained organisms, as well as the absence of contaminants. Moreover, both products tolerated the acidic conditions well, with encapsulation providing further protection for the microorganisms. A good tolerance to the simulated artificial intestinal conditions was also evidenced for both preparations.